Genome editing is a broadly applicable technology that enables targeted modification of DNA sequences in a wide range of cells and organisms. Recent widespread adoption of genome editing has been fueled by the simple programmability of CRISPR-Cas9 nucleases. Although CRISPR-Cas9 has democratized the ability to introduce targeted double-strand breaks (DSBs) and non-homologous end-joining (NHEJ)-mediated indel mutations, several key barriers still prevent this promising technology from reaching its full potential for research and therapeutic applications. The long-term goals of the proposed research program are: to develop innovative technologies that will have the largest impacts on use and application of genome editing; and to apply these advances to important problems in genetics, genomics, and biomedicine. The experiments described in this proposal are designed to address four major challenges that currently limit genome editing technology and its applications: (1) restrictions in the targeting range of CRISPR-Cas9 nucleases; (2) the inefficiency of nuclease-induced homology-directed repair (HDR) for creating precise alterations; (3) a limited understanding of the impacts of DNA sequence, chromatin state, and gene expression on the cleavage specificities of CRISPR-Cas9 nucleases; and (4) the lack of singularly specific and highly targetable Cas9- based nuclease platforms. For Challenge (1), a series of Cas9 variants with novel protospacer adjacent motif (PAM) specificities will be engineered that should broaden the targeting range of the platform. For Challenge (2), protein engineering will be used to create various synthetic reagents that recruit either HDR-promoting factors and/or DNA donor templates to the site of the nuclease-induced DSB. For Challenge (3), high- throughput technology for identifying CRISPR-Cas9-induced cleavage sites in genome-wide and unbiased fashion will be developed; this new method will then be used to generate large data sets that can serve to inform bioinformatics tools for better prediction of nuclease off target sites. For Challenge (4), the targeting range and size of a dimeric RNA-guided FokI-dCas9 nuclease platform recently described by our lab will be improved; in addition, a new class of monomeric CRISPR-Cas9 nucleases that are energetically poised to be active at on-target sites and inactive at even closely matched off-target sites will be engineered.